The proposed investigation will endeavor to extend our present knowledge of the mechanism of action of RNA polymerase. This is a very complex enzyme which catalyzes a series of complex steps leading to RNA synthesis and our aim is to construct model systems in order to more conveniently study certain of these steps. Having previously defined the length of the product site on RNA polymerase we now hope to extend these studies and determine the length of nascent RNA required to displace the sigma subunit. RNA polymerase catalyzes an active pyrophosphate reaction under appropriate experimental conditions. This is a partial reaction which does not require translocation. A comparison of pyrophosphate exchange with polymerization (in which translocation follows phosphodiester bond formation) should provide additional information with regard to the mechanism of translocation. We will also study reactions in which translocation is apparently reversed, i.e. pyrophosphorolysis and hydrolysis. Although a 3'-5' hydrolytic activity (exonucleolytic) has not been demonstrated, its presence, if shown, might suggest a proofreading function analogous to that shown for the bacterial DNA polymerases.